Chronic viral infections afflict more than 500 million people worldwide. Although altered stimulatory and inhibitory pathways contribute to T cell suppression during persistent viral infections in mice and humans, the underlying mechanisms are not fully understood. It should be noted that human persistent viruses, (including human immunodeficiency virus, hepatitis B and C viruses) are restricted to human and nonhuman primates and that the complexity of the immune system cannot be accurately recreated in vitro. Thus, the study of immune-regulation during chronic viral infections requires the use of appropriate whole organism viral systems. By using in vivo chronic lymphocytic choriomeningitis virus (LCMV) infection in mice, we and others have significantly contributed to understanding the roles and dynamics of several immune-regulators during chronic viral infections. In particular, we have shown that transforming growth factor beta (TGF?) production and signaling are sustained during persistent compared to acute LCMV infection. More recently, we have used temporally and cell-specifically controlled mouse genetics and observed that TGF? signaling in activated CD4 T cells is essential for host survival early after chronic (but not acut) LCMV infection and promotes viral replication at later stages. We also found that TGF? suppresses proliferation, terminal differentiation and (unexpectedly) a cytotoxic program in CD4 T cells early after infection while inhibiting CD8 T cell functions and accumulation at later stages. Importantly, initial data suggest that TGF? suppression of virus-specific cytotoxic CD4 T cells extends to HIV-infected patients. Understanding the cellular and molecular factors underlying the complex but critical role of TGF? and its crosstalk with contextual cytokines during an in vivo persistent viral infection is essential for manipulating this pathway to boost ani-viral defenses without causing immunopathology. To this end, in Aim #1 we propose to fully discern the temporal role of TGF? in modulating CD8 and CD4 T cell responses, immunopathology and viral control early versus late after chronic LCMV infection. We will also evaluate the role of TGF?3 and the TGF? activating molecule, integrin-?8, in immune responses and viral persistence. In Aim #2 we will determine the mechanisms underlying TGF? regulation of CD4 T cell responses by ablating SMAD-4 at different times after chronic LCMV infection and identifying SMAD-4 targeted gene promoters. We will also evaluate the role of transcription factors (TFs) that are putative SMAD-4 co-regulators and TFs that we found to be regulated by TGF? in CD4 T cells. Last, in Aim #3 we will examine whether interleukin-12 (IL-12) mediates the phenotypes induced by TGF? ablation during chronic LCMV infection, given that TGF? deficient CD4 T cells show enhanced IL-12 responsiveness. For that, we will block IL-12 in TGF?R sufficient and deficient mice and evaluate the consequences for T cell responses, immunopathology and viral control after persistent LCMV infection. These experiments will establish the foundation for future approaches aiming at enforcing IL-12 signaling to overcome TGF? suppression during chronic viral infection. In conclusion, our findings support the idea that TGF? plays vital roles during a chronic viral infection in vivo, influencing T cell responses, hos survival and viral control. While this suggests promising novel opportunities for immunotherapies, further understanding the dynamic activity of TGF? and the underlying molecular factors is necessary to effectively and safely manipulate this pathway and alleviate chronic viral diseases. Furthermore, given that LCMV is not only itself a pathogen of medical importance but it is also a prototypic member of the family arenaviridae, our work will help explaining how pathogenic arenaviruses suppress T cell responses to cause lethalhemorrhagic fevers in humans. Last, but not least, because TGF? is produced during several infections and T cells are central for anti-viral immunity, our work could have broad-reaching implications for multiple infectious diseases.